Ginseng is one of the most popular medicinal plants widely used for improving health. The root of ginseng has been consumed as a herbal tea in the traditional medicine and is currently used in a variety of products including candies, instant teas, and tonic drinks. Ginsenosides, which are glycosylated triterpene compounds contained in ginseng, may provide many positive effects on health. In particular, the ginsenosides have been known to have various pharmacological effects such as enhancement of the immune system and revitalization of the body function, and more than 40 different ginsenosides have been identified from the root of ginseng. However, the difficulty in large-scale production of the individual ginsenosides remains as a major obstacle to investigation of the efficacy of each ginsenoside, e.g., its therapeutic effect on specific diseases, and commercial use of the identified ginsenosides.
Ginsenosides are glycosylated dammarene-type tetracyclic triterpenes and can be classified into three different groups based on their aglycone structures: protopanaxadiol (PPD)-type ginsenosides, protopanaxatriol (PPT)-type ginsenosides and oleanolic acid-type ginsenosides. These three groups can be further classified based on the position and number of sugar moieties (aglycones) attached to the C-3, C-6 and C-20 positions of the rings in the chemical structure by a glycosidic bond. PPDs and PPTs have different hydroxylation patterns. While the PPDs have —OH groups at the C-3, C-12 and C-20 positions, the PPTs have —OH groups at the C-3, C-6, C-12 and C-20 positions. The PPDs and PPTs can be glycosylated with glucose or other sugars to be converted into various ginsenosides. The glycosylated PPD-type ginsenosides include ginsenosides Rb1, Rd, F2, Rg3, Rh2, compound K (C—K), Rb2, Rc, compound MC (C-MC), compound Y (C—Y), etc., and the glycosylated PPT-type ginsenosides include ginsenosides Rg1, Rh1, F1, Rf, Re, Rg2, etc.
The biosynthetic pathway of ginsenosides has been only partially identified. The ginsenosides are known to partially share the biosynthetic pathway with other triterpenes until oxidosqualene is synthesized by a series of condensation reactions of isopentenyl diphosphate and dimethylallyl diphosphate (DMADP) by the actions of IPP isomerase (IPI), GPP synthase (GPS), FPP synthase (FPS), squalene synthase (SS), and squalene epoxidase (SE) (Ajikumar et al. Science, 330, 70-74. 2010; Ro et al. Nature, 440, 940-943. 2006; Sun et al. BMC Genomics, 11, 262, 2010). Oxidosqualene is cyclized into dammarenediol-II by dammarenediol-II synthase (DS) which is a triterpene cyclase. Dammarenediol-II has hydroxyl groups at the C-3 and C-20 positions, and is converted to PPD by hydroxylation of the C-12 position by the p450 enzyme protopanaxadiol synthase (PPDS). PPDS can also be converted to PPT by hydroxylation at the C-6 position by another p450 protein, protopanaxatirol synthase (PPTS). PPD can be converted to various PPD-type ginsenosides by glycosylation at the C-3 and/or C-20 position(s), and PPT can be converted to various PPT-type ginsenosides by glycosylation at the C-6 and/or C-20 position(s).
Uridine diphosphate (UDP) glycosyltransferase (UGT) is an enzyme that catalyzes the transfer of a sugar moiety from a UDP-sugar to a wide variety of metabolites such as hormones and secondary metabolites. In general, UGT acts in the final step of the biosynthetic pathway in order to increase the solubility, stability, storage, biological activity or biological availability of metabolites. As recognized by the remarkable diversity of plant metabolites, each plant genome possesses hundreds of different UGTs. For example, a thale cress (Arabidopsis thaliana) plant model contains 107 UGTs that belong to 14 different groups (groups A to N) based on the amino acid sequences. However, although DS, PPDS, and PPTS have been reported as the enzymes involved in ginsenoside biosynthesis, little is known about whether UGT is involved in ginsenoside biosynthesis. Therefore, for production of specific ginsenosides, it is necessary to identify the UGTs, which use ginsenosides as substrates.
Different UGTs exhibit substrate specificity towards both sugar donors and sugar acceptors. For example, UGT78D2 transfers glucose from UDP-glucose to the C-3 position of a flavonol (kaempferol or quercetin) and an anthocyanin (cyanidin) in order to produce flavonol-3-O-glucosides and cyanidin-3-O-glucoside, respectively. It seems that such glycosylation is essential for in-vivo stability and storage of the compounds. On the other hand, UGT89C1 transfers rhanmnose from UDP-rhanmnose to the C-7 position of flavonol-3-O-glucoside in order to produce flavonol-3-O-glucoside-7-O-rhamnoside. Since UGT89C1 does not utilize UDP-glucose and anthocyanin-3-O-glucoside as substrates, it is known to exhibit different specificity towards the UDP-sugar and other acceptors from that of UGT78D2. As described, because different UGTs may have different substrate specificity and regioselectivity, it is necessary to investigate the substrate specificity and regioselectivity of the individual UGTs.